1. Field of the Invention
The present invention in the fields of molecular biology and medicine relates to methods for detecting specific sequences in double-stranded DNA samples and for detecting mutations and polymorphisms involving as little as one base change (Single Nucleotide Polymorphism—SNP) or additions to or deletions from the wild-type DNA sequence.
2. Description of the Background Art
Progress in human molecular and medical genetics depends on the efficient and accurate detection of mutations and sequence polymorphisms, the vast majority of which results from single base substitutions (as in SNPs) and small additions or deletions. Assays capable of detecting the presence of a particular mutation, SNP or mutant nucleic acid sequence in a sample are therefore of substantial importance in the prediction and diagnosis of disease, forensic medicine, epidemiology and public health. Such assays can be used, for example, to detect the presence of a mutant gene in an individual, allowing determination of the probability that the individual will suffer from a genetic disease and to detect the presence of an infectious agent in a patient. The ability to detect a mutation has taken on increasing importance in early detection of cancer or discovery of susceptibility to cancer with the discovery that discrete mutations in cellular oncogenes can result in activation of that oncogene leading to the transformation of that cell into a cancer cell and that mutations inactivating tumor suppressor genes are required steps in the process of tumorigenesis The detection of SNPs has assumed increased importance in the identification and localization (mapping) of genes, including those associated with human and animal diseases. Further, the continuing and dramatic increase in the number of SNPs of known location in the genome will allow genome wide scanning for identification of disease associated genes.
To realize the maximum potential benefits of this explosion of genetic information, both in research and in health care applications, and to increase the utility and applicability of mutation and SNP detection, will require improvements in current technologies, including increases in assay sensitivity and multiplexing ability and reductions in assay complexity and cost. The present invention is directed to methods of specific sequence, SNP and mutation detection embodying such improvements.
Most methods devised to attempt to detect genetic alterations comprising one or a few bases involve amplification of specific DNA regions by polymerase chain reaction (PCR). However, PCR amplification has severe limitations with respect to its utility in mutation and SNP detection:    1. PCR amplification is a relatively low fidelity process. Misincorporation during amplification is a particular problem in those detection methods that involve denaturation and annealing of PCR amplicons to form mutant:wild type heteroduplexes in which mutations and SNPs are revealed as mismatched or unpaired bases. Given the random nature of PCR errors, virtually all will be in such mismatches following annealing and will contribute to background signal. In gel based applications these error-containing molecules will generally not interfere. However, in high through put applications involving mismatch binding or mismatch cleaving, high background signals can greatly limit the utility of a method and frequently require that PCR fragments be kept relatively short.    2. PCR is subject to mispriming. Mispriming involves primer extension at non-target sites, which can occur even when only a relatively short portion of the 3′ end of a primer is transiently paired with some sequence in the target DNA. Mispriming can produce long single-stranded fragments which can adopt mismatch-containing secondary structure. Mispriming is also a major problem in those methods which utilize primer extension for SNP detection. These methods use oligonucleotides which are complementary to a region of target DNA immediately adjacent to the SNP or mutation to be genotyped such that the first nucleotide added by DNA polymerase to the 3′ end of the oligonucleotide will be complementary to and diagnostic for the SNP. Generally, these methods use specific nucleotide terminators (e.g., dideoxy or acyclo nucleotides) which are detectably labeled. Mispriming is such a problem with these methods that they generally require pre-amplification of the target region.    3. Some DNA regions are refractory to amplification. Because PCR requires denaturation of the target DNA, it provides the opportunity for the target DNA to adopt secondary structures, some of which may prevent primer annealing or extension.    4. PCR multiplexing potential is limited. The intricacies of primer design and the variability of PCR conditions depending on target and primer sequences coupled with the potential for interference between primer sets makes it unlikely that PCR will ever attain multiplexing levels as high as 100 fold, levels generally considered as the minimum desirable level for high through put SNP and mutation detection applications.
Therefore, a method of mutation/SNP that is not dependent on PCR amplification would have immediate and widespread utility both in research and health care. The present invention does not require PCR amplification.
Oligonucleotide Extension Assays
Oligonucleotide or primer extension (PE) assays allow detection of SNPs and mutations by addition of allele-specific nucleotides or nucleotide analogs to oligonucleotides, the 3′ end of which terminate at the nucleotide adjacent to the site in question. PE assays (Goelet , P et al., U.S. Pat. Nos. 5,888,819 and 6,004,744) are powerful methods for detecting known mutations and SNPs. PE requires PCR amplification of target DNA, removal of deoxynucleotide triphosphates (dNTPs) and PCR primers and addition of a third primer adjacent to the mutation or SNP site. Polymerase is then allowed to extend the specific primer by a single base by adding the nucleotide complementary to the mutation or SNP. Labeled terminator nucleotide analogs, e.g., dideoxy, arabinoside or acyclo nucleotides, are most commonly used. The precise nature of the nucleotide added is diagnostic of the allele in the target DNA. By using multiple differently labeled terminating nucleotide analogs, it is possible to genotype a given SNP or mutation site completely in a single reaction. Extension primers can contain 5′ adducts, such as biotin or specific oligonucleotide tails to allow immobilization and subsequent detection of the primers labeled by extension. Clearly, extension primers can be designed to allow detection of virtually all SNPs as well as deletion and addition mutations.
Primer extension assays suffer from the requirement for PCR amplification (due to problems with mispriming if extension primers are used with genomic DNA) and the consequent need to design three primers for each SNP or mutation site. Heretofore, and in clear distinction to the present invention, use of primer extension assays without target DNA denaturation and amplification have not been possible.
Oligonucleotide Ligation Assays (OLA)
The OLA method of SNP detection (Landegren, U et al., U.S. Pat. No. 4,988,617, Science 241:1077-1080 (1988), Methods ?? 9:84-90 (1996); Nickerson, D A et al., Proc Natl Acad Sci 87:8923-8927 (1990)) utilizes two oligonucleotide probes which are complementary to the target DNA flanking the SNP/mutation site. One oligonucleotide ends at the SNP site and the other ends at the nucleotide adjacent to the SNP site such that, when annealed to target DNA, the ends of both oligonucleotides are base-paired at the site of the SNP and can be joined (ligated) by DNA ligase. If there is a mismatch at the joint, i.e., if the end base in the oligonucleotide that covers the SNP/mutation is not complementary to the SNP/mutation, ligation cannot occur. Ligated products can be detected on gels without labeling by virtue of their increased length relative to the oligonucleotide probes.
To avoid the use of gels, OLA has been performed with probes which allow ligation product immobilization (Nickerson, D A et al., Proc Natl Acad Sci 87:8923-8927 (1990)). For example, one oligonucleotide probe can be prepared with a 5′ biotin adduct to allow immobilization to avidin- or streptavidin-coated plates or microspheres. This probe generally ends one nucleotide 5′ of the SNP. The other probe is detectably labeled, either internally or at the 3′ end. Detectable labels include fluorescent, radioactive and antigenic labels such as digoxigenin. In this example, the 5′ nucleotide of the detectably labeled probe would be complementary to one allele of the SNP or mutation. Target DNA must be denatured and the ligation oligonucleotides allowed to anneal. If the allele in question is present in the target DNA, ligation will occur and the labeled oligonucleotide will be linked to the biotinylated oligonucleotide such that label can be bound to avidin/streptavidin coated plates or microspheres. By combining two differently labeled oligonucleotides, each with their 5′ nucleotide complementary to a different allele of the SNP or mutation, it is possible to perform complete genotyping in a single assay (Samiotaki et al., Genomics 20:238-242 (1993)).
OLAs have also been used to detect changes in mononucleotide repeat sequence lengths (Zirvi, et al., Nucl Acids Res 27:e40 (1999)). The system is able to discriminate repeat sequences that vary by one nucleotide in sequences of up to 16 mononucleotide repeats. Notably, the above authors stated that: “The greatest source of error for analysis of mononucleotide repeat sequences, however, is the error generated during PCR amplification of microsatellite repeats.”
OLA has been used with flow cytometry and limited multiplexing (lannone, et al., Cytometry 39:131-140 (2000)). In this system, ligation products were immobilized to microspheres (Luminex) by means of a 25 base specific oligonucleotide “tail”. Target DNA was PCR-amplified prior to ligation. Simultaneous genotyping of 9 SNPs was demonstrated.
By virtue of their precision and specificity OLAs have enjoyed widespread application in mutation and SNP detection. However, and in clear distinction to the present invention, OLAs absolutely require denaturation of target DNA and have not been successful without amplification of target sequences.
RecA
RecA is a bacterial protein involved in DNA repair and genetic recombination and has been best characterized in E. coli. RecA is the key player in the process of genetic recombination, in particular in the search and recognition of sequence homology and the initial strand exchange process. RecA can catalyze strand exchange in the-test tube. Recombination is initiated when multiple RecA molecules coat a stretch of single-stranded DNA (ssDNA) to form what is known as a RecA filament. This filament, in the presence of ATP, searches for homologous sequences in double-stranded DNA (dsDNA). When homology is located, a three stranded (D-loop) structure is formed wherein the RecA filament DNA is paired with the complementary strand of the duplex.
RecA homology searching is extremely precise and RecA has been used to facilitate screening of plasmid libraries for plasmids containing specific sequences (Rigas et al., Proc Natl Acad Sci USA. 83:9591-9595 (1986)). In this approach, biotinylated ssDNA probes are reacted with RecA to form RecA filaments. The filaments are used for homology searching in circular plasmid DNA. When the probes are removed by binding to avidin, those plasmids containing sequences homologous to the probes are isolated by virtue of the triple stranded (D-loop) structures formed by the RecA filament and the plasmid duplex. Stabilization of these structures requires the use of adenosine 5′-[γ-thio]triphosphate (ATP[γ-S]) in place of ATP. ATP[γ-S] allows homology searching by RecA, but is non-hydrolyzable and thus does not allow RecA to dissociate from the triple stranded structure.
RecA has also been used, in a variety of applications, to facilitate the mapping and/or isolation of specific DNA regions from bacterial and human genomic DNA (Ferrin, L J, et al., Science 254:1494-1497 (1991); Ferrin, L J, et al., Nature Genetics 6:379-383 (1994); Ferrin, L J et al., Proc Natl Acad Sci 95:2152-2157 (1998), Sena et al., U.S. Pat. Nos. 5,273,881 and 5,670,316; Sena and Zarling, Nature Genetics 3:365-371 (1993)). In one of these applications (Ferrin et al., 1991, supra; 1994, supra; U.S. Pat. No. 5,707,811), RecA is used in conjunction with restriction enzymes (sequence-specific double strand DNA endonucleases) to allow isolation or identification of specific DNA fragments. RecA filaments are prepared and reacted with genomic DNA under conditions that allow triple strand (D-loop) structure formation. The DNA is then treated with either a restriction endonuclease or a modification methylase (methylase action transfers a methyl group to the specific recognition sequence of a specific restriction endonuclease, thus protecting the sequence from endonuclease digestion). The presence of the RecA filament in the triple strand structure prevents digestion or methylation.
In a more recently developed application (Ferrin et al., 1998, supra and U.S. Pat. No. 5,707,811;), specific RecA filaments were used to protect restriction endonuclease generated “sticky ends” from being filled in by DNA polymerase such that, upon removal of the RecA filaments, specific fragments can be cloned into plasmid vectors. In this application (??), genomic DNA is digested with one or more restriction enzymes that produce recessed 3′ ends. A specific fragment from this digestion is protected by triple strand structure formation with a pair of RecA filaments. The recessed 3′ ends of the remaining fragments are then filled in with a polymerase. The polymerase is removed or inactivated, the RecA, filament is removed and the specific fragment cloned by virtue of its sticky ends.
RecA has been used in association with DNA ligase to label specific DNA fragments (Fujiwara, J et al., Nucl Acids Res 26:5728-5733 (1998)). Oligonucleotides are designed to allow the 3′ end to form a double-stranded region by folding back on a portion of itself (hairpin), RecA is then used to coat the remaining single-stranded 3′ region and the resulting RecA filament used to perform homology searching. When a terminus of the target DNA is complementary to the single-stranded portion of the oligonucleotide, ligation can covalently link the oligonucleotide, which can be labeled at the 5′ end with a detectable label, to the target DNA to allow detection or isolation of specific target DNA sequences without denaturation of the target DNA.
Formation of RecA catalyzed double D-loops has been used to identify and isolate specific DNA regions from ds DNA (Sena et al., supra; Sena and Zarling, supra). This method requires relatively long DNA probes (>78 nucleotides), and complementarity between the probes and double D-loops in order to provide for a stable structure. The above documents note the possibility of introducing a detectable label into the probe by oligonucleotide extension with DNA polymerase. Importantly, this method is suited only for detection of specific sequences in a target DNA but is of no use in detecting mutations or SNPs—both of which are objectives of the present invention.
In summary, no applications of RecA have heretofore been proposed that allow the detection of mutations or SNPs or the identification of sequences which differ from a wild type sequences by only one or a few nucleotides.
All statements as to the date or representation as to the contents of documents cited herein is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.